Project 1: Vinculin is required for cell polarization, migration and extracellular matrix remodeling in 3D collagen Ingo Thievessen Vinculin is an F-actin binding protein enriched in integrin-based adhesions to the extracellular matrix (ECM). While studies in two-dimensional (2D) tissue culture models have suggested that vinculin negatively regulates cell migration by promoting cytoskeleton-ECM coupling to strengthen and stabilize adhesions, its role in regulating cell migration in more physiological, three-dimensional (3D) environments is unclear. To address the role of vinculin in 3D cell migration, we analyzed morphodynamics, migration and ECM-remodeling of primary murine embryonic fibroblasts (MEF) with cre/loxP-mediated vinculin gene disruption in 3D collagen I cultures. We found that vinculin promoteds 3D cell migration by increasing directional persistence. Vinculin was required for persistent cell protrusion, cell elongation and stable cell orientation in 3D collagen, but was dispensable for lamellipodia formation, suggesting that vinculin- mediated cell adhesion to the ECM is required to convert actin-based cell protrusion into persistent cell shape change and migration. Consistent with this, vinculin was required for efficient traction force generation in 3D collagen without affecting myosin II activity, and promoted 3D collagen fiber alignment and macroscopical gel contraction. Our results suggest that vinculin promotes directionally persistent cell migration and tension-dependent ECM-remodeling in complex 3D environments by increasing cell-ECM adhesion and traction force generation. This work was done in collaboration with Ben Fabry, Rudolf Oldenbourg and Eric Betzig, and a manuscript describing these results was published in FASEB this past year Project 2:Specific protein-interactions regulate the three-dimensional nanoscale organization of vinculin within focal adhesions Lindsay Case Previous studies have shown that FAs have a conserved stratified nanoscale structure, with an integrin signaling layer (ISL) 10-20 nm from the plasma membrane, an actin regulatory layer (ARL) 100 nm from the membrane that extends into the stress fiber, and a force transduction layer (FTL) that spans these two layers. Vinculin is an FA protein that functions in signaling, force transduction, and regulation of the actin cytoskeleton. Vinculin has at least 10 binding partners distributed throughout the FA including paxillin in the ISL, talin in the FTL, and actin in the ARL. We hypothesize that vinculin interacts with distinct binding partners within distinct FA layers to regulate its activation and mediate its functional specificity. To test this, we utilized point mutants to perturb specific protein interactions and assayed their nanoscale localization, activation state, and binding stability in FAs. Our results suggest a model in which inactive vinculin is recruited to the ISL near the plasma membrane in FA by a weak interaction with paxillin. We speculate that this localization puts vinculin in proximity of multiple ligands that promote activation, which allows a shift to the FTL and ARL where interactions of activated vinculin with talin and actin promote FA stabilization. We are assaying additional vinculin mutants to provide further evidence for this model. This work was done in collaboration with Mike Davidson, Sharon Campbell, and Harald Hess, and a Manuscript describing this work and a related review of the literature were published in Nature Cell Biol. Project 3: Active organization of integrins in focal adhesions Vinay Swaminathan Integrins are transmembrane ECM receptors that link the extra-cellular matrix (ECM) and the cytoskeleton and play a crucial role in the immune response, cell migration and tissue morphogenesis. ECM-engaged integrins cluster together with proteins that mediate their signaling functions and linkage to the cytoskeleton to form focal adhesions that grow and turn over in an actin and myosin II dependent manner. How actin and myosin mediate the clustering and organization of integrins during activation, focal adhesion growth and turnover is not known. We utilized fluorescence emission anisotropy imaging of cells expressing GFP-tagged integrins to analyze the evolution of integrin organization during focal adhesion dynamics in migrating fibroblasts and to test the role of integrin activation and actomyosin contractility in this process. By employing specific perturbations we are attempting to understand how the association of focal adhesion components and the acto-myosin machinery affect the organization of integrins during the formation of mature adhesions. This work was done in collaboration with Timothy Springer, Satyajit Mayor, David Baker, and Tomomi Tani, and 2 manuscripts are in preparation Project 4: A novel actin-adhesion structure requiring the formin FMN2 positions the nucleus and protects it from DNA damage during confined migration Colleen T. Skau Force transmission to the nucleus has been shown to mechanically initiate signaling events and control chromatin organization, but the contribution of the actin cytoskeleton versus nuclear lamins in this process is poorly understood. Similarly, coupling the nucleus to the cytoskeleton can control the position of the nucleus within the cell. We examined the interplay between actin, adhesions, and the nucleus in fibroblasts using fluorescence microscopy. We identified novel adhesion structures located underneath the nucleus termed subnuclear adhesions that are compositionally and dynamically distinct from the canonical focal adhesions at the leading edge. Subnuclear adhesions are nucleated along an existing actin fiber, in contrast to leading edge adhesions. We show that the actin fibers connecting two subnuclear adhesions can control nuclear shape by physically impinging on the nucleus. These subnuclear fibers have elevated levels of the IIB isoform of myosin but reduced levels of &#945;-actinin as compared with dorsal stress fibers, and are less dependent on the contractile activity of myosin than dorsal stress fibers. Furthermore, subnuclear fibers are independent of the Arp2/3 complex but dependent on activity of the formin FMN2. We find that FMN2 localizes as thin dynamic fibrils underneath the nucleus in cells plated on 2 dimensional substrates, partially co-localizes with subnuclear actin bundles, and in a cup-like organization around the rear of the nucleus in cells in 3D collagen gels. Critically, FMN2 is essential for both subnuclear actin and adhesions; loss of FMN2 eliminates both structures. These cells exhibit defects in nuclear positioning and show increased double-stranded breaks in DNA upon mechanical compression while migrating. We therefore propose a unique role for the novel actin-adhesion system generated by FMN2: The nucleus-associated actin-adhesion system functions to protect the nucleus from mechanical damage during confined migration as well as control the position of the nucleus in migrating cells. This is the first demonstration of an actin-adhesion system responsible for protecting the nucleus from physical damage in migrating fibroblasts.